2-methylthioadenosine-5&#39;-monophosphate

ABSTRACT

2-METHYLTHIOADENOSINE-5&#39;&#39;-MONOPHOSPHATE AND THE AMMONIUM, SODIUM AND POTASSIUM SALTSA THEREOF AND PHARMACEUTICAL COMPOSITIONS AND METHODS OF INHIBITING OR PREVENTING THROMBUS FORMATION WITH THESE COMPOUNDS

United States Patent 3,781,274 Z-METHYLTHIOADENOSINE- 5'-MONOPHOSPHATE Mary Helen Maguire and Geoffrey Gough, Mosman, and

Frank Michal, Lane Cove, New South Wales, Australia, assignors to The University of Sydney, New South Wales, Australia No Drawing. Original application Dec. 8, 1969, Ser. No. 883,354, now Patent No. 3,678,162. Divided and this application Jan. 21, 1972, Ser. No. 219,912

Int. Cl. C07d 51/54 US. Cl. 260211.5 R 2 Claims ABSTRACT OF THE DISCLOSURE 2-methylthioadenosine-S-monophosphate and the ammonium, sodium and potassium salts thereof and pharmaceutical compositions and methods of inhibiting or preventing thrombus formation with these compounds.

This is a division of application Ser. No. 883,354, filed Dec. 8, 1969, now Pat. No. 3,678,162.

This invention relates to new chemical compounds, namely 2-methylthioadenosine-5-monophosphate and the ammonium, sodium and potassium salts thereof. This invention also relates to pharmaceutical compositions and methods of inhibiting or preventing thrombus formation with these compounds.

It has been found that Z-methylthioadenosine-5'-monophosphate is useful for the inhibition or prevention of thrombus formation in, for example, post-operative situations, cases of heart implants with pacemakers and artificial valves, organ transplants, atherosclerosis, venousthrombotic states and conditions where platelet adhesion is excessive.

The activity of Z-methylthioadenosine 5 monophosphate is demonstrated by the following procedures.

Z-methylthioadenosine 5' monophosphate at 0.5 ,uM causes a 50% inhibition of adeonsine diphosphate (ADP)-induced aggregation of sheep blood platelets in 3,781,274 Patented Dec. 25, 1973 "Ice platelet-rich plasma in vitro. Similarly, a inhibition of the ADP-mediated aggregation of dog and human platelets is caused by 1.3 #M and 8.6 ,uM 2-methylthioadenosine-5'-monophosphate. The inhibition produced is of long duration. The technique used for the quantitative measurement of platelet aggregation is described in the article by M. H. Maguire and F. Michal, Nature, 217, 571 (1968). 2-methylthioadenosine-5'-monophosphate reduces the stickiness of platelets in vivo, demonstrated by the following procedures.

2-methylthioadenosine-5'-phoshate (250-500 ,ug./kg.) was administered to dogs anaesthetised with Nembutal or chloralose and readministered at intervals of to minutes. Blood was sampled at hourly intervals, and the response to ADP of the platelets in platelet-rich plasma isolated from the blood samples was studied. A significant reduction in the ADP-induced aggregation of platelets was observed, and the platelet aggregates formed disaggregated more rapidly than normal. Results were obtained from experiments on seven dogs.

Thrombi can be induced in exposed cerebral cortex arterioles in the rat by low voltage electrical stimulation applied across the vessel wall. Z-methylthioadenosine-S'- monophosphate when injected intravenously in doses of -200 ug/kg. significantly increased the time a thrombus took to form, compared to the time in which control thrombi formed in the absence of the drug, for a period of approximately 60 minutes after each injection. Results were obtained from experiments on seven different animals.

At doses of 2000 ,ugjkg. injected intravenously into anaesthetized rats and guinea pigs, 2-methylthioadenosine- 5'-monophosphate does not produce arrhythmias or heart block and does not cause substantial depression of arterial blood pressure.

Doses of 75 mg./kilo-gram administered intravenously in mice showed no toxic effects.

Z-methylthioadenosine-5'-monophosphate may be prepared for example from Z-methylthioadenosine by either of the following processes:

NHa NHa NH N N i I N 1 CHaS N CHSL \N N 011s onwo HO8CH\N N 3 N N 2 O NC(CH2)2OPOCH2 0 noom l H+ on on j \G/ c on ' our orr,

NH2 N N\ N I ons OHS I I S \N N a N N 11+ H H o-1 oom 0 taro-Poem o o- H H c CH1 CH8 droxy groups of 2-methyl-thioadenosine are protected bytreating with acetone in the presence of an acid condensing agent such as for example p-toluene sulphonic acid to give 2-methylthio-2',3'-isopropylideneadenosine.

The 2-methylthio-2,3'-isopropyl-ideneadenosine is then reacted with fi-cyanoethylphosphate in a solvent such as pyridine, using dicyclohexylcarbodiimide as a condensing agent.

The B-cyanoethyl and isopropylidene groups are removed With, respectively, alkali (for example lithium hydroxide) and acid (low pH) to give Z-methylthioadenosine-'-monophosphate. This product may be purified by treating with barium hydroxide to give the barium salt, converting the barium salt to the ammonium salt by ionexchange chromatography, purifying the ammonium salt by column chromatography and then converting to the free acid, 2-methylthioadenosine-5'-monophosphate, by elution from an anion exchange column with formic acid.

Alternatively, the 2 methylthio-2',3'-isopropylideneadenosine is treated with phosphorus oxychloride in the presence of trimethyl or triethyl phosphate. Excess phosphorus oxychloride is hydrolysed with aqueous lithium hydroxide. Lithium phosphate is removed, the solution is heated at pH 2 for 30 minutes and Z-methylthioadenosine- 5-monophosphate is isolated as the barium salt, and purified as described in the first procedure.

The free acid may be converted to its sodium or potassium salt by passage through a sodium or potassium cation exchange column and evaporation to dryness.

For therapeutic use, Z-methylthio-adenosine-S'-monophosphate is preferably administered in the form of a pharmaceutical composition comprising the active ingredient in combination with a pharmaceutical carrier. These pharmaceutical compositions also fall within the scope of the present invention. Advantageously, the compositions can be made up in a dosage unit form appropriate to the desired mode of administration, which may be intravenous or intramuscular.

The pharmaceutical carrier may be for example, pyrogen free normal saline.

The following examples illustrate the invention and are not intended to limit the scope thereof.

EXAMPLE 1 2-methylthio-2',3'-O-isopropylidene adenosine (a) Pure 2-methylthioadenosine (2 g.) and p-toluene sulphonic acid (12.8 g.) were suspended in dry acetone (450 ml.) and the mixture was stirred for 90 minutes with a Vibromix. After 10 min. a clear solution was obtained, after 30 min. crystals separated. The mixture was poured into 350 ml. of ice water containing 13.4 g. of NaHCO and the solution obtained was evaporated to dryness at 30-35".

The residue was extracted by repeated heating of the reaction product of the acetone (8x 500 ml.) and decanting of the acetone extract. Evaporation of acetone left an oil which was dissolved in 500 ml. of boiling H O, charcoaled and the solution evaporated to ml. in vacuo at Crystals separated and were filtered (1.55 g.) and crystallised again by evaporation from an aqueous methanolic solution. The aqueous mother liquors yielded another 200 mg. and recrystallisation of the combined products from H O gave needless (1.4 g.) M.P. 177178.5, chromatographically homogeneous. Analysis:

req. (percent): C, 47.6; H, 5.38; N, 19.8. Found (percent): C, 47.66; H, 5.28; N, 19.70.

It has been found advantageous to use a dimethoxypropane:acetone (1:10) mixture in the initial acetonation step, to use a longer reaction time, and to extract the product from the NaHCO evaporation residue with chloroform instead of acetone.

(b) 2-methylthioadenosine (2.5 g.) and p-toluene sulphonic acid (16 g.) were suspended in dry acetone (750 ml.) containing 2,2-dimethoxypropane (40 ml.) and the mixture was stirred for 4 hours, then poured into ice water (400 ml.) containing NaHCO 18.2 g.). The solution was evaporated to dryness and the residue was dried in vacuo over NaOH. The residue was then extracted with boiling chloroform (3 ml.). Evaporation of chloroform left an oil which was crystallised from hot water yielding 2.2 g. (78% of theory) of the pure product.

2-methylthioadenosine-5-monophosphoric acid (a) 1.06 g. (3 moles) of 2-methylthio-2',3'-O-isopropylidene adenosine in 25 ml. pyridine was allowed to stand at room temperature with 12 ml. of 1 mmoles/ml. stock solution of fl-cyanoethyl phosphate and 6 g. of dicyclohexylcarbodiimide. The reaction was terminated after 24 hr. by the addition of 5 ml. of water and the resultant mixture evaporated to dryness. ml. of 0.4 N lithium hydroxide solution was added to the residue and the mixture was refluxed for 1 hr. to remove the cyanoethyl protecting group. It was then cooled and filtered, and the filtrate was passed through a column of Bio-Rad AG 50WX4 cation exchange resin (H+ form) to replace lithium by hydrogen ions. The acidic efiluent was left at room temperature overnight to effect cleavage of the isopropylidene protecting group. It was then concentrated to about 150 ml. and its pH adjusted to 7.5 with barium hydroxide solution. Barium phosphate which precipitated was centrifuged off and the supernatant was treated with two volumes of ethanol and allowed to stand overnight. The crude nucleotide barium salt which then settled out was collected by centrifugation, washed with ethanol, acetone and ether and dried, yielding 1.9 g.

300 mg. of crude barium salt was converted to the ammonium salt by passage through a column of cation exchange resin. It was then applied in a small volume of isopropanol: 0.25 M aqueous ammonium bicarbonate (2:1) to a 2.5 x 35 cm. column of cellulose packed in the same solvent. Elution with this solvent yielded a pure fraction of the desired nucleotide as the ammonium salt. Ammonium bicarbonate from the solvent was removed by repeated evaporation of water and the residue was converted from the ammonium salt form to the free acid by absorbing it on a column of Bio-Rad AG1-X4 anion exchange resin, washing the column well with water, then eluting it with 4 N formic acid. The fractions of the effluent which contained the nucleotide were concentrated and dried to yield 2-methyl-thioadenosine-5'-monophosphoric acid as a white solid mg., approx. 64% yield from the isopropylidene derivative).

The solid was recrystallised twice from water to give white needles, M.P. 192-195 C. (decomposition). Analysis: C11H16N5O7PS'H2O req. (percent): C, 32.12; H, 4.41; N, 17.03; P, 7.53. Found (percent): C, 31.77; H, 4.42; N, 16.94; P, 7.45.

The isolation of 2-methylthioadenosine-S-phosphate may also be achieved advantageously without proceeding via the barium salt as described hereunder in (b).

(b) 1.06 g. of Z-methylthio 2,3' O isopropylidene adenosine was treated as described above up to the step involving reflux with 0.4 N lithium hydroxide. The reaction mixture was then filtered and the filtrate was adjusted to pH 1.5 with 89% phosphoric acid. The acid solution was heated at 70-80 C. for 2 hrs., then treated with dilute lithium hydroxide solution until the pH reaches 9. Precipitated lithium phosphate was filtered off. The filtrate was evaporated to c. 30 ml. and passed through 2.0 x 30 cm. column of Bio-Rad AG 50W-X4 (H+) which retained the nucleotide. The column was washed with 500 ml. water then eluted with 300 ml. of 1 M NH OH. The eluate was evaporated to 15 ml. then strongly acidified with formic acid. Z-methylthioadenosine-5'-monophosphate began to crystallise out immediately.

The mixture was refrigerated overnight then filtered to give 899 mg. of chromatographically homogeneous white needles (73% yield).

A further 266 mg. of crude product was isolated from the mother liquor by absorption on a column of Bio-Rad AG2-X8 and elution with 4 N formic acid. This was recrystallised once from water to give the pure product (197 mg., total yield 89% (c) 1.06 g. (3 mmoles) 2-methylthio-2',3'-O-isopropylidene adenosine was dissolved in a solution of 2.73 ml. (30 mmoles) phosphoryl chloride and 1.43 ml. (6 mmoles) of tri-n-butylamine in 5 ml. dry trimethyl phosphate at 0 C. Atter 72 hrs. at 0 C., the reaction mixture was poured into water (250 ml.), the pH adjusted to 1.5 with LiOH solution, and the resulting sticky mass stirred at 70 C. for 1 hr. during which time most of it dissolved. The solution was adjusted to pH '9 with LiOH and the precipitate of lithium phosphate filtered off. The filtrate was passed through a 2.5 x 35 cm. column of Bio- Rad AG 50W-X4 (H+) which retained the nucleotide. The column was washed with 300 ml. water then eluted with 1 M NH OH. The alkaline eluate was evaporated to c. 50 m1. then applied to a 3.2 x 28 cm. column of Bio-Rad AG2-X8 (for-mate form) and followed by 500 ml. water. Elution was then begun with 4 M HCOOH. 15 ml. fractions were collected. Z-methylthioadenosine-S'- monophosphate emerged in fractions 34-43. These fractions were pooled and incorporated to give 260.3 mg. white crystalline product (21% yield).

6 EXAMPLE 2 Z-methylthioadenosine 5' monophosphate in water is passed through a sodium cation exchange resin and the solvent is removed by evaporation in vacuo to give the sodium salt of Z-methylthioadenosine 5 monophosphate.

Similarly, the potassium salt of Z-methylthioadenosine- 5' monophosphate is prepared by passing 2-methylthioadenosine 5' monophosphate in water through a potassium cation exchange resin and removing the solvent by evaporation in vacuo.

What is claimed is:

1. A compound selected from the group consisting of 2 methylthioadenosine 5' monophosphate and the ammonium, sodium and potassium salts thereof.

2. A compound according to claim 1, said compound being 2-methylthioadenosine-5'-monophosphate.

References Cited UNITED STATES PATENTS 3,225,029 12/1965 Yamaoka 260211.5 R 3,380,996 4/1968 Honjo et al. 260211.5 R 3,586,606 6/1971 Nakayama et al. 260211.5 R

JOHNNIE R. BROWN, Primary Examiner US. Cl. X.R. 424- 

